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Part:BBa_K2592007

Designed by: Janice Law   Group: iGEM18_Hong_Kong_HKU   (2018-09-23)


Strand 5 for in-vivo synthesis of Nano Drug Carrier-AS1411 (NDC-as)

This part encodes a component strand of our originally designed Nano Drug Carrier-AS1411 (NDC-as). The strand-encoding region is followed by an HIV-terminator binding site (HTBS). This component strand can be expressed in the presence of HIV reverse transcriptase and murine leukaemia virus reverse transciptase inside E. coli DH5alpha (Elbaz, 2016).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 13
    Illegal NheI site found at 36
    Illegal NheI site found at 83
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



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Introduction

Biology

Part structure

This biobrick is part of a DNA nanostructure production system we named ETHERNO (E. coli-synthesized Therapeutic Nanostructures). Each of the biobrick submitted encodes a single-stranded DNA of specific sequence. These ssDNA synthesized can then be extracted for assembly of our originally designed therapeutic nanostructure.

In each biobrick, the component strand encoding region is followed by an HTBS, a stemloop for binding of HIV reverse transcriptase and B0054, a strong terminator. For our design to function, these three structural components must be together as a basic part. This part design is modified from a previously described method of ssDNA synthesis using this HTBS sequence and B0054 terminator.[1] Each component strand encoding region is originally designed, with component strand sequence generated by 3D nanostructure simulation in the software TIAMAT. [2]

For nanostructures composed of multiple DNA strands of different sequences, the BioBricks required follow the same basic structure as shown in Fig. 1.

Figure 1. ETHERNO workflow

Therapeutic DNA nanostructure

Design principles: safety, efficient cell entry, flexibility, stability

In this project, 2 DNA nanostructures, namely Nano Drug Carrier (NDC) and Nano Drug Carrier-AS1411 (NDC-AS) for breast cancer therapy were designed and tested. Each nanostructure is made up of 5 single-stranded DNA synthesized using 5 different BioBricks. The 2 nanostructures have 2 componant strands in common, so a total of 8 BioBricks were made and submitted.

Our Nano Drug Carriers are composed entirely of DNA, which is non-toxic and degradable inside human cells. The Nano Drug Carriers are designed as tetrahedrons to facilitate cell entry. Previous studies have shown that three-dimensional DNA nanostructure enter mammalian cells more efficiently than two-dimensional AS1411 or linear structures. [3]Tetrahedron is chosen because we consider it the simplest three-dimensional structure, which can be easily assembled from just a few DNA strands. Building the Nano Drug Carrier with separate DNA strands means that much flexibility is allowed for functional modifications. Functional DNA sequences can be conveniently added to the 4 vertices of the tetrahedron to achieve oligonucleotide delivery or cell antigen binding, enhancing the effect of the drug. Three-dimensional structures composed of double stranded DNA have been shown to be stable in extracellular compartment, making them ideal as drug carriers.[4]

Figure 2. NDC
Our NDC (Fig. 2) for breast cancer therapy can be conveniently assembled by annealing 5 single-stranded DNA: 4 core strands forming a tetrahedron and 1 strand (Strand 5) to be displaced by target miRNAs, miR21 and miR217 inside breast cancer cells.[5] Part of Strand 5 is a displacement toehold complimentary to miR21. The 5' segment next to the toehold is complimentary to another target, miR217. The 3' segment binds complimentarily to 5' end of Strand 1. Breast cancer drug, doxorubicin (Dox) can be loaded onto the tetrahedron by DNA intercalation.[6] Therapeutic functions are expected to be exerted in two ways: cancer miRNA down-regulation by the displaced strand and intracellular release of doxorubicin.


Figure 3. NDC-AS
Inspired by the Human Practices interview with molecular pathologist Dr. Lau, NDC-AS (Fig. 3) was designed for more cell-specific drug delivery. AS1411, a previously characterized DNA aptamer against nucleolin was incorporated into Strand 2 of NDC. [7]As nucleolin is overexpressed in breast cancer cells, NDC-as was expected to preferentially enter cancer cells and demonstrate better entry and cancer cell cytotoxicity than NDC.[8]The complimentary region between the tetrahedral base and the strand to be displaced was also extended to increase displacement specificity. Extension was done by adding TCG/AGC repeats to facilitate doxorubicin intercalation.

Characterization

NDC-AS

To test the feasibility of our designs, chemically synthesized DNA oligos of the sequences generated by Tiamat were used to assemble the NDC-AS. DNA-21 and DNA-217, which are DNA equivalents of miR21 and miR217 were initially used as the inputs, before using RNA. Native polyacrylamide gel electrophoresis (PAGE) was used to visualize the component strands and the assembled NDC-AS.



Figure 4.PAGE (8%) image of NDC-AS component single strands and assembled NDC-AS

After assembly, component strands were seen to have formed complexed too large to be run through the gel PAGE (Fig.4). These large complexes close to the loading well were expected to be successfully assembled NDC that could not pass through the gel due to its large 3D structure, with a tetrahedral base of around 16nm per edge. With extended complimetary region between Strand 1-AS and Strand 5-AS.



Thioflavin detection of AS1411 formation

To test if the AS1411 aptamer on NDC-AS was successfully formed, we incubated 1μM of our nanostructures with 10μM Thioflavin T at room temperature for 10 minutes. Thioflavin T is a selective sensor of DNA G-quadruplex. Thioflavin gives off fluorescence upon binding to G-quadruplex due to its restricted rotation and enforced planarization [1]. High fluorescence signal after incubation of NDC-AS with Thioflavin T would indicate successful formation of AS1411, which has a G-quadruplex structure [2]. Thioflavin T was incubated with water only as control.

NDC-AS, after incubation with Thioflavin T, emitted significantly higher fluorescence than NDC without AS1411. This shows that G-quadruplex was formed in NDC-AS.

Parts application

The collected single-stranded DNA (ssDNA) were suspended in water which contained leftovers of chemicals used in extraction. Because of this, these ssDNA could not be easily visualized on PAGE, as performed above for NDC assembled from chemically synthesized ssDNA. None the less, we tried to amplify the ssDNA extracted using primers that overlapped with the component ssDNA sequences. We expected to get back bands of the expected ssDNA sizes as shown in Fig.1 and Fig.6 on PAGE. We also annealed the strands synthesized by ETHERNO, to see if large complexes similar to NDC and NDC-AS as shown in Fig.1 and Fig.6 could be produced.

Products of ETHERNO

Expected bands of Strand-1, Strand 1-AS, Strand 5 and Strand 5-AS can be seen in Fig.20a, which means these 4 strands were present in the ssDNA extracted from ETHERNO. Other strands could not be amplified here, may be due to problematic primer design. Designing primers for these nanostructure component strands was challenging due to the complicated secondary structures formed. Since these extracted ssDNA were able to form large structures that could not pass through PAGE.(Fig. 20b), we moved on to look for any successfully assembled NDC and NDC-AS under transmission electron micrpscopy.

TEM of NDC made by ETHERNO
TEM of NDC-AS made by ETHERNO

Cloning

Colony PCR to check presence of parts

References

  1. ↑ Elbaz, J., Yin, P. & Voigt, C.A. (2016, April 19). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nat Commun. 7:11179.
  2. ↑ Williams, S., Lund, K., Lin, C., Wonka, P., Lindsay, S., & Yan, H. (2009). Tiamat: A three-dimensional editing tool for complex DNA structures. In Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) (Vol. 5347 LNCS, pp. 90-101). (Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics); Vol. 5347 LNCS). DOI: 10.1007/978-3-642-03076-5_8
  3. ↑ Xia, Z., Wang, P., Liu, X., Liu, T., Yan, Y., Yan, J....He, D. (2016, March 8). Tumor-penetrating peptide-modified DNA tetrahedron for targeting drug delivery. Biochemistry, 55(9),1326-1331.
  4. ↑ Kumar, V., Palazzolo, S., Bayda, S., Corona, G., Toffoli, G. & Rizzolio F. (2016). DNA Nanotechnology for Cancer Therapy. Theranostics, 6(5), 710-725.
  5. ↑ Singh, R. & Mo, Y. (2013, March 1). Role of microRNAs in breast cancer. Cancer Biol Ther, 14(3), 201–212.
  6. ↑ Sun, G. & Gu, Z. (2015, January 26). Engineering DNA Scaffolds for Delivery of Anticancer Therapeutics. Biomaterials Science, 3(7), 1018-1024.
  7. ↑ Bates, P.J., Laber, D.A., Miller, D.M., Thomas, S.D. & Trent, J.O. (2009, June). Discovery and Development of the G-rich Oligonucleotide AS1411 as a Novel Treatment for Cancer. Experimental and Molecular Pathology, 86(3), 151–164.
  8. ↑ Fonseca, N.A., Rodrigues, A.S., Rodrigues-Santos, P., Alves, V., Gregório, A.C., Valério-Fernandes, A.... Moreira, J.N.(2015, November). Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination. Biomaterials, 69, 76-88.
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